The present invention relates to the detection of Chlamydia and to the diagnosis, treatment and prevention of Chlamydia infections in animals.
Chlamydiae are obligate intracellular bacterial pathogens with a unique biphasic life cycle. They appear as two distinct cellular types, a small dense cell or elementary body (EB) that is enclosed in a rigid bacterial cell wall, and a larger metabolically active reticulate body (RB). The EB is resistant to physical disruption and is infectious, whereas the RB is more fragile and only exists inside cells. The Chlamydia life cycle begins with the attachment of the EB form to the host cell which is followed by endocytosis into a nascent vacuole, also called an xe2x80x9cinclusion membranexe2x80x9d. After EB attachment and entry, replication of the EB form produces RB forms that continue to grow within the vacuole. By 72 hour post-infection, this growth phase is terminated when the RBs condense, and reorganize back to EBs. The lysis of the host cell results in release of EBs to infect new host cells. The difficulties in working with Chlamydiae center on the obligate intracellular requirement for growth and the fact that no adequate genetic engineering methods have been developed for this organism.
The genus Chlamydia includes two species that are primarily associated with human disease: C. trachomatis and C. pneumoniae. C. trachomatis causes trachoma, an eye disease that is the leading cause of preventable infectious blindness worldwide with an estimated 500 million cases of active trachoma worldwide. C. trachomatis also causes a sexually transmitted chlamydial disease which is very common worldwide. C. trachomatis also causes lymphogranuloma venereum, a debilitating systemic disease characterized by lymphatic gland swelling. The most serious sequelae of chlamydial genital infections of females include salpingitis, pelvic inflammatory disease, and ectopic pregnancy. In the U.S. alone, it is estimated that over 4 million new sexually transmitted C. trachomatis infections occurred in 1990, leading to over four billion dollars in direct and indirect medical expenses. The World Health Organization estimates that 89 million new cases of genital Chlamydia occurred worldwide in 1995 (Peeling and Brunham, 1996).
C. pneumoniae causes respiratory diseases including so called walking pneumonia, a low-grade disease such that the infected person frequently fails to obtain treatment and remains in the community as an active, infectious carrier. C. pneumoniae is currently of interest because of its strong epidemiological association with coronary artery disease, and there is also some evidence to link it with multiple sclerosis.
Of the other disease-causing species of Chlamydia, Chlamydia psittaci and Chlamydia pecorum are primarily pathogens of wild and domestic animals, but these species may infect humans accidentally. C. psittaci is acquired through respiratory droplet infection and is considered an occupational health hazard for bird fanciers and poultry workers.
There is tremendous interest in the identification of candidate antigens for protection against chlamydial disease. While a prior infection with C. trachomatis will protect against a subsequent challenge by the same strain, indicating a protective component that stimulates the host immune response, most serious chlamydial diseases are exacerbated by an overaggressive anti-chlamydial immune response. Antigens recognized in the context of an infection appear to elicit a protective response whereas immunization with purified, killed (EB form) Chlamydia results in an immunopathological response. Therefore for the purposes of vaccine development, one needs to find epitopes that confer protection, but do not contribute to pathology. It is an object of this invention to provide Chlamydia polypeptides for use as vaccines that induce a protective immune response without inducing the pathological response caused by the antigens associated with the EB form of Chlamydia. Such immunostimulatory peptides will be useful in the treatment, as well as in the diagnosis, detection and prevention of Chlamydial infections.
The present invention includes the use of Chlamydia proteins that show enhanced expression in the reticulate body (RB) stage relative to the elementary body (EB) stage of the Chlamydia life cycle. These proteins are not present at detectable levels in the EB form using current immunological techniques and are thus said to be xe2x80x9cinfection-specific.xe2x80x9d Certain of these infection-specific proteins are found in the inclusion membrane of the infected cell, and so have been termed xe2x80x9cIncxe2x80x9d proteins. These include the IncA, IncB, and IncC proteins of Chlamydia as described in the present disclosure. The genes that encode the IncA, IncB and IncC proteins are referred to as incA, incB and incC respectively. Other proteins of Chlamydia described herein have also been shown by the inventors to be infection-specific, but are not known to be incorporated into the inclusion membrane; these include the p242, TroA, and TroB proteins. The TroA and TroB proteins have been so named because they resemble the Tro proteins of Treponema pallidum, which are thought to form part of an ABC transport system.
The inventors have shown that the infection-specific Chlamydia proteins of the disclosure are recognized by convalescent antisera (i.e., antisera taken from an animal that has recovered from a Chlamydia infection) but are not recognized by antisera against the killed EB form of Chlamydia. Thus, the proteins are expressed only during active chlamydial infection and are therefore useful as protective antigens. These infection-specific proteins may be used to confer a protective immune response without inducing a pathological effect. Additionally, immuno-fluorescence microscopy and immunoblotting with antisera demonstrated that the infection-specific proteins are present in Chlamydia-infected HeLa cells, but are undetectable in purified EBs and absent in uninfected HeLa cells.
Immunofluorescense microscopy reveals that IncA, IncB and IncC are localized to the inclusion membrane of infected HeLa cells. Reverse-transcription polymerase chain reactions (RT-PCR), northern hybridization data, and restriction analysis revealed that the incB and incC genes are closely linked and transcribed in an operon. RT-PCR, restriction analysis and sequential Southern hybridizations of incA then incC to the same filter provided evidence that incA is separated from the incB and incC operon by about 110 kb. The C. trachomatis Tro genes are not closely linked with the p242 gene.
The present invention includes the nucleotide and amino acid sequences for certain infection-specific proteins from Chlamydia. These proteins are p242, TroA, and TroB from C. trachomatis, and the IncB, and IncC proteins from C. psittaci. The scope of the invention includes fragments of these proteins that may be used in a vaccine preparation or that may be used in a method of detecting Chlamydia antibodies. Such fragments may be, for example, 5, 10, 15, 20, 25, or 30 contiguous amino acids in length. They may even encompass the entire protein.
More specifically, the present invention encompasses the purified infection-specific proteins having amino acid sequences as shown in SEQ ID NOS: 2, 4, 6, 10, and 12, amino acid sequences that differ from such sequences by one or more conservative amino acid substitutions, and amino acid sequences that show at least 75% sequence identity with such amino acid sequences.
Then invention also includes isolated nucleic acid molecules that encode a protein as described in the above paragraph, including isolated nucleic acid molecules with nucleotide sequences as shown in SEQ ID NOS: 1,3, 5, 9, and 11.
The present invention also includes a vaccine or immunostimulatory preparation directed against the reticulate body (RB) form of Chlamydia comprising one or more purified infection-specific peptides (or portions or fragments thereof, or peptides showing sequence similarity to a portion of such a peptide). Such peptide fragments may be, for example, 5, 10, 15, 20, 25, or 30 contiguous amino acids in length, of the sequence shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, or 18. Peptides used in such a vaccine may even encompass the entire purified:!peptide of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, or 18, a peptide that differs from such a peptide by one or more conservative amino acid substitutions, or a peptide having at least 75% sequence identity with such a peptide. Such vaccine preparations may contain one or more pharmaceutically acceptable excipients, adjuvants or diluents.
The invention additionally encompasses methods for making a vaccine, comprising combining a pharmaceutically acceptable excipient with a peptide described herein. Also included is a method of vaccination comprising administering a vaccine as described herein to a mammal.
The present invention also provides a method for the diagnostic use of the disclosed purified infection-specific peptides, for instance by use in a diagnostic assay to detect the presence of infection-specific antibodies in a medical specimen, in which antibodies bind to the Chlamydia peptide and indicate that the subject from which the specimen was removed was previously exposed to Chlamydia. Such a method may comprise: (i) supplying a biological sample, such as blood from an animal, that is suspected to contain infection-specific anti-Chlamydia antibody, (ii) contacting the sample with at least one infection-specific Chlamydia peptide described herein, such that a reaction between the peptide and the infection-specific anti-Chlamydia antibody gives rise to a detectable effect, such as a chromogenic conversion; and (iii) detecting this detectable effect.
The present invention also provides a method of using antibodies that bind specifically with the disclosed proteins for detection of infection-specific Chlamydia antigen, indicating the presence of Chlamydia in the RB stage as distinct from the EB stage. For instance, the relevant infection-specific antibodies may be used to provide specific binding in an Enzyme Linked Immunosorbant Assay (ELISA) or other immunological assay wherein the antibody Fc portion is linked to a chromogenic, fluorescent or radioactive molecule and the Fab portion specifically interacts with, and binds to, an infection-specific protein. Such a method may comprise: (i) supplying a biological sample from an animal suspected to contain an infection-specific Chlamydia antigen, and (ii) contacting the sample with at least one infection-specific anti-Chlamydia antibody, such that a reaction between the antibody and the infection-specific Chlamydia protein gives rise to a detectable effect; and (iii) detecting this detectable effect.
Other aspects of the present invention include the use of probes and primers derived from the nucleotide sequences that encode infection-specific peptides, to detect the presence of Chlamydia nucleic acids in medical specimens. Such probes and primers may be nucleotide fragments, of, for example, 15, 20, 25, 30 or 40 contiguous nucleotides of the sequence shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, or 17.
An additional aspect of the invention is a method of treating a Chlamydia infection by directing a therapeutic agent against a specific target, where the target is chosen from an infection specific protein of Chlamydia, a gene that encodes an infection-specific protein of Chlamydia, and an RNA transcript that encodes an infection-specific protein of Chlamydia, wherein the therapeutic agent interacts with said target to affect a reduction in pathology.
These and other aspects of the invention will become more apparent from the following description.
SEQ ID NO:1 shows a nucleic acid sequence encoding the p242 C. trachomatis protein, with deduced primary amino acid sequence also shown.
SEQ ID NO:2 shows the amino acid sequence of the p242 C. trachomatis protein.
SEQ ID NO:3 shows a nucleic acid sequence encoding the TroA C. trachomatis protein, with deduced primary amino acid sequence also shown.
SEQ ID NO:4 shows the amino acid sequence of the TroA C. trachomatis protein.
SEQ ID NO:5 shows a nucleic acid sequence encoding the TroB C. trachomatis protein, with deduced primary amino acid sequence also shown.
SEQ ID NO:6 shows the amino acid sequence of the TroB C. trachomatis protein.
SEQ ID NO:7 shows a nucleic acid sequence encoding the IncA C. psittaci protein, with deduced primary amino acid sequence also shown.
SEQ ID NO:8 shows the amino acid sequence of the IncA C. psittaci protein.
SEQ ID NO:9 shows a nucleic acid sequence encoding the IncB C. psittaci protein, with deduced primary amino acid sequence also shown.
SEQ ID NO:10 shows the amino acid sequence of the IncB C. psittaci protein.
SEQ ID NO:11 shows a nucleic acid sequence encoding the IncC C. psittaci protein, with deduced primary amino acid sequence also shown.
SEQ ID NO:12 shows the amino acid sequence of the IncC C. psittaci protein.
SEQ ID NO:13 shows a nucleic acid sequence encoding the IncA C. trachomatis protein, with deduced primary amino acid sequence also shown.
SEQ ID NO:14 shows the amino acid sequence of the IncA C. trachomatis protein.
SEQ ID NO:15 shows a nucleic acid sequence encoding the IncB C. trachomatis protein, with deduced primary amino acid sequence also shown.
SEQ ID NO:16 shows the amino acid sequence of the IncB C. trachomatis protein.
SEQ ID NO:17 shows a nucleic acid sequence encoding the IncC C. trachomatis protein, with deduced primary amino acid sequence also shown.
SEQ ID NO: 18 shows the amino acid sequence of the IncC C. trachomatis protein.
SEQ ID NO: 19 shows the upstream oligonucleotide used to amplify the C. psittaci incC ORF.
SEQ ID NO:20 shows the downstream oligonucleotide used to amplify the C. psittaci incC ORF.
SEQ ID NO:21 shows the upstream oligonucleotide used to amplify the C. psittaci incB ORF.
SEQ ID NO:22 shows the downstream oligonucleotide used to amplify the C. psittaci incB ORF.
SEQ ID NO:23 shows the upstream oligonucleotide used to amplify the C. psittaci incA ORF.
SEQ ID NO:24 shows the downstream oligonucleotide used to amplify the C. psittaci incA ORF.
Particular terms and phrases used herein have the meanings set forth below.
xe2x80x9cEBxe2x80x9d refers to the Elementary Body, an environmentally refractile and largely metabolically dormant form of Chlamydia that is infectious and is presented as a small dense body enclosed by a bacterial cell wall.
xe2x80x9cRBxe2x80x9d refers to the Reticulate Body, a metabolically active form of Chlamydia that is not infectious, and exists only within a host cell being very fragile, often branched, and appearing larger and less dense that the EB.
xe2x80x9cInfection-specificxe2x80x9d refers to a protein that shows enhanced expression in the RB form of Chlamydia compared to the EB form. Infection-specific proteins are not necessarily absent from the EB form, but they are significantly more common in the RB form than in the EB form.
xe2x80x9cinfection-specific antibodyxe2x80x9d is an antibody that binds specifically to an infection-specific protein.
xe2x80x9cBiological samplexe2x80x9d refers to any sample of biological origin including, but not limited to a blood sample, a plasma sample, a mucosal smear or a tissue sample.
xe2x80x9cIsolatedxe2x80x9d An isolated nucleic acid has been substantially separated or purified away from other nucleic acid sequences in the cell of the organism in which the nucleic acid naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA. The term xe2x80x9cisolatedxe2x80x9d thus encompasses nucleic acids purified by standard nucleic acid purification methods. The term also embraces nucleic acids prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
xe2x80x9cProbesxe2x80x9d and xe2x80x9cprimers.xe2x80x9d Nucleic acid probes and primers may readily be prepared based on the nucleic acid sequences provided by this invention. A xe2x80x9cprobexe2x80x9d comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
xe2x80x9cPrimersxe2x80x9d are short nucleic acids, typically DNA oligonucleotides 15 nucleotides or more in length, which are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.
Probes and primers as used in the present invention typically comprise at least 15 nucleotides of the nucleic acid sequences that are shown to encode infection-specific proteins. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 30 or 40 consecutive nucleotides of the disclosed nucleic acid sequences.
Methods for preparing and using probes and primers are well known in the art and are described in, for example Sambrook et al. (1989); Ausubel et al., (1987); and Innis et al., (1990). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).
xe2x80x9cConservative amino acid substitutionsxe2x80x9d are those substitutions that, least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions.
Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalnyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.
xe2x80x9cSequence identityxe2x80x9d The similarity between two nucleic acid sequences, or two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences are. Variants of naturally occurring infection-specific peptides useful in the present invention are typically characterized by possession of at least 50% sequence identity counted over the full length alignment with the amino acid sequence of a naturally occurring infection-specific peptide when aligned using BLAST 2.0.1 (Altschul et al., 1997). For comparisons of amino acid sequences of greater than about 30 amino acids, the BLAST 2 analysis is employed using the blastp program set to default perameters (open gap=11, extension gap=1 penalty, gapxc3x97dropoff=50, expect=10, word size=3, filter on), and using the default BLOSUM62 matrix (gap existence cost=11, per residue gap cost=1 lambda ratio =0.85). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix (gap existence cost=9, per residue gap cost=1, lambda ratio=0.87). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at http//www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine sequence identity using this program is available at http://www.ncbi.nlm.nih.gov/BLAST/blast help.html.
Similarly, when comparing nucleotides, blastn may be used with default settings (rewards for match=1, penalty for mismatch=xe2x88x922, open gap=5. extension gap=2 penalty, gapxc3x97dropoff=50, expect=10, word size=11, filter on), with the default BLOSUM62 matrix (as above). Variants of naturally occurring infection-specific nucleic acid sequences useful in the present invention are typically characterized by possession of at least 50% sequence identity counted over the full length alignment with the nucleic acid sequence of a naturally occurring infection-specific ORF when aligned using BLAST 2.0.1. Useful nucleic acids may show even greater percentage identity, and may, for example, possess at least 55%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity naturally occurring infection-specific ORF.
xe2x80x9cOperably linkedxe2x80x9d A first nucleic acid sequence is xe2x80x9coperablyxe2x80x9d linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
xe2x80x9cRecombinantxe2x80x9d A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
xe2x80x9cStringent Conditionsxe2x80x9d Stringent conditions, in the context of nucleic acid hybridization, are sequence-dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5 degrees to 20 degrees lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al. (1989), pages 9.49-9.55. Typical high stringency hybridization conditions (using radiolabeled probes to hybridize to nucleic acids immobilized on a nitrocellulose filter) may include, for example, wash conditions of 0xc3x97SSC, 0.5% SDS at a wash temperature of 68xc2x0 C.
When referring to a probe or primer, the term xe2x80x9cspecific for (a target sequence)xe2x80x9d indicates that the probe or primer hybridizes under high-stringency conditions substantially only to the target sequence in a given sample comprising the target sequence.
xe2x80x9cPurifiedxe2x80x9d A purified peptide is a peptide that has been extracted from the cellular environment and separated from substantially all other cellular peptides. As used herein, the term peptide includes peptides, polypeptides and proteins. In certain embodiments, a purified peptide is a preparation in which the subject peptide comprises 50% or more of the protein content of the preparation. For certain uses, such as vaccine preparations, even greater purity may be preferable.
xe2x80x9cImmunostimulatory peptidexe2x80x9d as used herein refers to a peptide that is capable of stimulating a humoral or antibody-mediated immune response when inoculated into an animal.
xe2x80x9cVaccinexe2x80x9d A vaccine is a composition containing at least one immunostimulatory peptide which may be inoculated into an animal with the intention of producing a protective immunological reaction against a certain antigen. The antigen to be protected against may be, for instance, an infection-specific antigen of Chlamydia.
Isolation of IncA, IncB and IncC
Bacterial strains. Chlamydia (C. psittaci strain GPIC or C. trachomatis LGV-434, ser. L2) was cultivated in HeLa 229 cells using standard methods (Caldwell et al., 1981). Purified Chlamydia were obtained using Renografin (E. R. Squibb and Sons, Inc., Princeton, N.J.) density gradient centrifugation. Escherichia coli DH5xcex1 (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) was used as the host strain for transformations with recombinant DNA. E. coli XL1-Blue MRFxe2x80x2 (Stratagene, La Jolla, Calif.) was used as the host strain for infection with lambda ZAPII phage vector. E. coli SOLR (Stratagene) was used as the host strain for infection with in vivo excised filamentous lambda ZAPII.
Antisera. MBP (Maltose Binding Protein)-Inc fusion proteins were used as antigens for the production of mono-specific antibody reagents in Hartley strain guinea-pigs. The protein was diluted to 100 xcexcg/mlxe2x88x921 sterile saline and mixed with the Ribi Trivalent Adjuvant (Ribi Immunochem.). The antigen/adjuvant emulsion was administered to anaesthetized guinea-pigs using a procedure provided by the manufacturer. Sera were collected 14 days after secondary and tertiary imnmunizations. Control antisera were produced by immunizing guinea-pigs with adjuvant alone, or with adjuvant plus purified maltose-binding protein.
Convalescent guinea-pig antisera, antisera against live EBs, and antisera against formalin-fixed EBs were produced using standard methods (Rockey and Rosquist, 1994 and. Rockey et al., 1995).
C. psittaci library construction and screening. For the incB and incC genes, C. psittaci strain GPIC DNA was extracted using a genomic DNA extraction kit (Qiagen) with one modification; dithiothreitol (5 mM) was added to the suspension buffer to assist EB lysis. DNA was partially digested with Tsp509I and ligated to EcoRI digested xcex-ZAPII phage arms (Stratagene). The ligation was packaged in vitro with Gigapack extracts accordinglito the manufacturer""s instructions (Stratagene). Recombinant phage were plated on E. coli XL-1 Blue at densities of approximately 104 PFU/150mm (diameter) plate. Following a nine hour incubation to allow development of the plaques, the plates were sequentially overlaid with nitrocellulose disks and the resulting lifts were processed for immunoblotting with convalescent antisera and antisera to fixed EBs. Of approximately 8,000 plaques, 18 had reactivity with the convalescent sera but not sera generated against EBs. One of these was subcloned into pBluescript SK(xe2x88x92) phagmid by in vitro excision in the E. coli SOLR strain (Stratagene) and designated pBS200-7.
For the incA gene, genomic DNA from C. psittaci strain GPIC was partially digested with Sau3A, size-selected (2-8 kb) by electrophoresis through low-melting-temperature agarose, and blunt-ended with T4 DNA polymerase. This DNA was ligated to an EcoR1/Not1 adapter (Life Technologies), kinased, and ligated to EcoR1-digested Lambda ZAP II vector (Stratagene Cloning Systems). Recombinants were packaged (Lambda Gigapack Gold, Stratagene) and used to infect E. coli XL1-Blue (Stratagene). Plaques were allowed to develop for 4 h at 37xc2x0 C. Nitrocellulose filters laden with 10 mM IPTG (U.S. Biochemical Corp.) were placed onto the plaques and incubated for an additional 4 h at 37xc2x0 C. These filters were removed and placed into a blocking solution consisting of PBS (150 mM NaCl, 10 mM NaPO4, pH7.2) plus 0.1% Tween-20 (TPBS) and 2% BSA-TPBS. Filters were incubated for 1 h, rinsed twice in TPBS, and incubated overnight in convalescent-guinea-pig sera diluted 1:100 in BSA-TPBS. After three washes in TPBS, the filters were incubated for 1 h in 125I-staphylococcal protein A (New England Nuclear) diluted to approx. 124 nCimlxe2x88x921 in BSA-TPBS. Filters were again washed three times in TPBS and positive plaques were detected by exposure of the dried filters to autoradiography film overnight at room temperature. Positive clones were picked and plaque-purified. pBluescript-SK-plasmids containing the chlamydial genes of interest were recovered from the purified bacteriophage using ExAssist filamentous bacteriophages (Stratagene).
Identification of antigens recognized by convalescent antisera. Recombinant plaques were identified that showed reactivity with convalescent (anti-RB) antisera, but not with anti-EB serum. The purified recombinant phage were converted into pBluescriptII SK plasmid by in vivo excision and recircularization and these recombinant DNAs were used to transform E. coli. SDS-PAGE and immunoblot analysis of lysates of these recombinant E. coli showed that each expressed one or more proteins that reacted with convalescent antisera but not with the EB serum.
DNA Cloning and fusion protein production. The plasmid pJC2 contain""s a 5.0 kb EcoRI GPIC genomic fragment cloned into the pZEro2.1 vector (Invitrogen). To construct pJC2, the incC ORF sequence was 32P-radiolabeled using random priming (Gibco-BRL) and used to probe EcoRI cut GPIC genomic DNA fragments separated by agarose gel electrophoresis. Fragments in the size range of the positive signal were excised from the gel and purified by Gene-Clean (Bio101). The gel-purified fragments were used in a ligation along with EcoRI-digested pZEro2.1. Kanamycin resistant colonies were screened by colony hybridization with radiolabeled incC.
MBP fusions of the five ORFs present in pJC2 were produced using the pMAL-C2 vector (New England Biolabs). The reading frame of incC, with the exception of the first four codons, was amplified using Pwo polymerase (Boehringer Mannheim) and pBS200-7 as the template. The upstream and downstream oligonucleotides for this amplification were
5xe2x80x2-AGAACCGATTTAACTCCAGGCG-3xe2x80x2 (SEQ ID NO: 19) and
5xe2x80x2-GCGCGGATCCTTAATGTCCGGTAGGCCTAG-3xe2x80x2 (SEQ ID NO: 20), respectively.
The vector was digested with XmnI and BamHI, and the amplication product was digested with BamHI. Ligation of these products resulted in an in-frame fusion between the malE gene in the vector and the incC reading frame from pBS200-7. The stop codon for this construction is provided by the insert. Following ligation, the products were transformed into E. coli strain HD5xcex1. The resulting fusion protein (MBP/IncC) was overexpressed and purified by maltose affinity chromatography using an amylose resin supplied by New England Biolabs.
The same approach was used for production of the MBP/IncB fusion protein. The sequence encoding the N-terminal 101 amino acids of the IncB ORF was PCR amplified using the oligonucleotides
5xe2x80x2-ATGTCAACAACACCAGCATCTTC-3xe2x80x2 (SEQ ID NO: 21) and
5xe2x80x2-GCGCGGATCCTTAATTAGTGCCTTCTGGATTAGG-3xe2x80x2 (SEQ ID NO: 22).
The purified MBP/IncB and MBP/IncC fusion proteins were used as antigen for the production of monospecific antibody in Hartley strain guinea-pigs by standard methods (Rockey et al., 1995). Inserts in each construct were confirmed by DNA sequencing.
For IncA, a maltose-binding protein/IncA fusion protein was produced using the pMAL-C2 vector system from New England Biolabs. The reading frame of incA shown in FIG. 1, with the exception of the initiator ATG, the incA ORF was amplified using Vent DNA polymemase (New England Biolabs) and plasmid pGP17 as template. The upstream and downstream ogligo-nucleotides for this amplification were
5xe2x80x2-CGCAGTACTGTATCCACAGACAAC-3xe2x80x2 (SEQ ID NO: 23) and
5xe2x80x2-GTCGGATCCGAGAAACTCTCCATGCC-3xe2x80x2 (SEQ ID NO: 24), respectively. The vector was digested with XmnI and BamHI, and the amplification product was digested with ScaI and BamHI. Ligation of these products resulted in an in-frame fusion between the malE gene in the vector and the incA reading frame from pGP17. The stop codon for this construction is provide by the insert. Following ligation, the products were transformed into E. coli strain DH5xcex1. The resulting fusion protein (MBP/IncA) was overexpressed and purified by maltose affinity chromatography using amylose resin (New England Biolabs).
MBP/IncA was used as antigen for the production of monospecific antibody reagents in Hartley strain guinea-pigs.
DNA sequencing and sequence analysis. The pBS200-7 and pJC2 genomic clones as well as the MBP fusions were sequenced with the Taq DyeDeoxy Terminator Cycle Sequencing Kit (Perkin Elmer/Applied Biosystems Division). Several internal primers were designed to sequence further into the cloned inserts. Sequence assembly was performed using AssemblyLIGN software and sequence analysis was performed with MacVector software (International. Biotechnologies Incorporated). Hydrophilicity profiles were determined using the Kyte-Doolittle scale (Kyte and Doolittle, 1982) with a window of 7. Deduced amino acid sequences were compared with the database using the BLAST program (on default settings) available from the National Center for Biotechnology Information on the world wide web. The entire nucleotide sequence of the pJC2 insert was deposited in the GenBank/EMBL Nucleotide Sequence Data Library, under accession number AF017105.
For incA, nucleotide sequencing was conducted using the Sequences system, (US Biochemical) with the M13 forward and reverse primers, and internal primers synthesized on an Milligen/Biosearch Cyclone Plus DNA synthesizer. Computer analyses were conducted using the MacVector Sequence Analysis Software (International Biotechnologies Incorporated). Hydrophilicity profiles were determined using the Kyte-Doolittle scale (Kyte and Doolittle, 1982) with a window of 7. Secondary-structure predictions were generated using a combination of the Chou-Fasman and Robson-Garnier methods (Robson and Suzuki, 1976; Chou and Fasman, 1978). Deduced amino acid sequences were compared with those in the EMBL and GenBank databases using the BLASTP program available from the National Center for Biotechnology Information.
Electrophoresis and immunoblotting. Polyacrylamide gel electrophoresis (PAGE) was conducted using standard methods (Rockey and Rosquist, 1994). Immunoblotting was performed using standard methods (Rockey et al., 1995).
Immunofluorescence studies. Chlamydiae grown in HeLa cells on sterile glass coverslips were fixed for microscopy one of two ways. Cells were either incubated in methanol for 5 minutes, or in the combination fixative periodate-lysine-paraformaldephyde (PLP) for three hours at room temperature followed by permeabilization with 0.05% saponin (Brown and Farquhar, 1989). Immunostaining of the fixed coverslips was performed according to standard methods (Rockey et al., 1995) and visualized under a Nikon Microphot FXA microscope using the 63xc3x97 objective and oil immersion.
RT-PCR analysis. RNA for RT-PCR analysis was extracted from approximately 2xc3x971014 C. psittaci-infected cells. A Qiagen column was used for extraction and purification according to the manufacturer""s instructions (Qiagen). RQ1 RNase DNase (Promega) was used to ensure removal of contaminating genomic DNA. cDNA was prepared by incubating 1.5 xcexcg of RNA, 2.5 xcexcM of the reverse oligonucleotide primer, and AMV reverse transcriptase (Promega) for 1 hour at 42xc2x0 C. in sodium pyrophosphate buffer, according to the manufacturer""s instructions. PCR reactions were carried out using 1 xcexcl of the cDNA reaction, 1.25 xcexcM of each oligonucleotide primer, and Pwo polymerase (Boehringer Mannheim). Each RT-PCR reaction was accompanied by a positive control reaction that utilized the same primer set and 10 ng of C. psittaci genomic DNA, and a negative control reaction in which 1 xcexcl of the same RNA preparation was used as template in the PCR reaction. A control primer set located within the incC gene was also used as an RT-PCR control.
Identification of incA, incB and incC genes of C. trachomatis. The nucleotide sequence information obtained for the incA, incB and incC of C. psittaci (above) was used, with standard methods, to identify the inc gene orthologues of C. trachomatis. Probes were made that corresponded to the 3xe2x80x2 and 5xe2x80x2 ends of the C. psittaci inc open reading frames. Standard PCR amplification (as above) was used, with the C. trachomatis genome as a template, to amplify the corresponding C. trachomatis nucleotide sequence. The amplified DNA was then sequenced, using standard methods.
2. Isolation of p242, TroA and TroB
Bacterial strains. C. trachomatis LGV-434, serotype L12, was cultivated in HeLa 229 cells using standard methods (Caldwell et al., 1981). Purified chlamydiae were obtained using Renografin (E. R. Squibb and Sons Inc., Princton, N.J.) density gradient centrifugation (Hackstadt et al., 1992). Escherichia coli DH5xcex1 (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) was used as the host strain for transformations with recombinant DNA. E. coli XL1-Blue MRFxe2x80x2 (Stratagene, La Jolla, Calif.) was used as the host strain for infection with lambda ZAPII phage vector. E. coli SOLR (Stratagene) was used as the host strain for infection with in vivo excised filamentous lambda ZAPII.
Antisera. Two Cynomolgus monkeys (Macaca fasicularis) were anaesthetized and infected urethrally with C. trachomatis EBs. Each monkey was infected twice and allowed to recover between infections. Symptoms of infection were monitored over time. Antisera from infected monkeys were tested for reactivity to Chlamydia by ELISA (Su et al., 1990).
Sera were collected every two weeks and anti-chlamydial titers were determined. These animals showed mild clinical signs of disease which cleared spontaneously. A second challenge was then administered. Sera were collected from these animals and used to probe a C. trachomatis expression library as discussed below. As a control, Guinea Pigs were immunized with killed C. trachomatis of the EB form. Sera from these animals were obtained and also used to probe the C. trachomatis expression library.
C. trachomatis library construction and immunoscreening. A C. trachomatis genomic library was constructed with the lambda ZAPII vector as described above for C. psittaci. Approximately 15,000 plaques were plated, transferred to nitrocellulose filters (Schleicher and Schuell, Keene, N. H.) in duplicate, and probed with the monkey convalescent antiserum and with Guinea Pig serum against killed EBs (Bannantine et al., 1998). Plaques that reacted only with the monkey convalescent antisera were selected for further study.
Identification of antigens recognized by convalescent antisera. Four positive recombinant plaques were identified that showed reactivity with convalescent antisera but not with anti-EB serum. The purified recombinant phage were converted into pBluescriptII SK plasmid by in vivo excision and recircularization and these recombinant DNAs (pCt1, pCt2, pCt3 and pCt4) were used to transform E. coli. SDS-PAGE and immunoblot analysis of lysates of these recombinant E. coli showed that each expressed one or more proteins that reacted with convalescent (anti-RB) antisera but not with the anti-EB antiserum. Two of the recombinants clones, pCt2 and pCt3, expressed an identical 19.9 kDa protein (p242). The pCt4 recombinant expressed two different proteins of approximately 32 kDa each that are strongly recognized by convalescent antisera (TroA and TroB).
Sequence analysis of pCt1, 2, and 3 revealed overlapping inserts with only one open reading frame (ORF) common in all three. This ORF encodes an approximately 19.9 kDa protein (p242) that shows no similarity to other known proteins. The nucleotide sequence encoding C. trachomatis p242, and the amino acid sequence of the protein are shown in SEQ ID NOS: 1 and 2, respectively.
The insert in pCt4 contains two complete ORFs which code for two proteins, each of approximately 32 kDa (TroA and TroB) that show some homology with proteins from Treponema pallidum. The nucleotide sequences encoding the 32 kDa proteins (TroA and TroB) and the amino acid sequences of these proteins are shown in SEQ ID NOS: 3, 4, 5, and 6.
The present invention includes the nucleotide and amino acid sequences for certain infection-specific proteins from Chlamydia. These proteins are p242, TroA, and TroB from C. trachomatis, and the IncB, and IncC proteins from C. psittaci. The scope of the invention includes fragments of these proteins that may be used in a vaccine preparation or that may be used in a method of detecting Chlamydia antibodies. Such fragments may be, for example, 5, 10, 15, 20, 25, or 30 contiguous amino acids in length, or may even encompass the entire protein.
The present invention also encompasses the use of infection-specific proteins of Chlamydia, and the use of nucleotides encoding such proteins. Infection-specific proteins include the IncA, IncB and IncC proteins of C. psittaci, the IncA, IncB and IncC proteins of C. trachomatis, and the TroA, TroB, and p242 proteins of C. trachomatis. The inventors have shown that these proteins are infection-specific by using immunological techniques such as immuno-fluorescence microscopy and immunoblotting.
The present invention includes a vaccine against chlamydial infections comprising infection-specific proteins or fragments of these proteins or proteins that are homologous or show substantial sequence similarity to these proteins. In one embodiment, one or more purified infection-specific proteins may be mixed with a pharmaceutically acceptable excipient to produce a vaccine that stimulates a protective immunological response in an animal. In one embodiment the vaccine may be administered intra-muscularly or subcutaneously or intravenously. In another embodiment, the vaccine may be administered by inoculation into or onto the mucous membranes of the subject animal. For example, the vaccine may be administered urethrally or genitally as a liquid or in the form of a pessary. In another embodiment, it may be administered to the mucosa of the lungs as a spray or vapor suspension.
Since at least thee amino acids are required to produce an antigenic epitope, the vaccine should comprise at least three consecutive amino acids, preferably at least five consecutive amino acids, and may comprise at least 10, 15, 25, 30, 40, or 45 consecutive amino acids of the infection-specific proteins as shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, and 18.
The vaccine of the invention may be used to inoculate potential animal targets of any of the chlamydial diseases including those caused by C. psittaci. C. trachomatis, C. pneumoniae or C. pecorum. Indeed the vaccine of the invention may be used to inoculate animals against any disease that shows immunological cross-protection as a result of exposure to infection-specific Chlamydia antigen.
Vaccines of the present invention can include effective amounts of immunological adjuvants known to enhance an immune response (e.g., alum). The protein or polypeptide is present in the vaccine in an amount sufficient to induce a protective immune response whether through humoral or cell mediated pathways or through both. Such a response protects the immunized animal against chlamydial infections specifically by raising an immune response against the Reticulate Body form of Chlamydia. Protective antibodies may be elicited by a series of two or three doses of the antigenic vaccine given about two weeks apart.
The present invention also teaches a method of making a vaccine against chlamydial infections. The method of making the vaccine comprises providing a pure (or substantially pure) infection-specific chlamydial peptide or portion thereof, and mixing the peptide with a pharmacologically acceptable excipient or adjuvant. Adjuvants may include commonly used compounds such as alum. Additionally, the vaccines may be formulated using a peptide according to the present invention together with a pharmaceutically acceptable excipient such as water, saline, dextrose and glycerol. The vaccines may also include auxiliary substances such as emulsifying agents and pH buffers. Doses of the vaccine administered will vary depending on the antigenicity of the particular peptide or peptide combination employed in the vaccine and characteristics of the animal or human patient to be vaccinated.
The infection-specific vaccine of the invention is directed towards not only C. psittaci, but against all forms of Chlamydia including C. pneumoniae, C. trachomatis and C. pecorum, and the vaccine may comprise not just peptides derived from C. psittaci, but also orthologous peptides and fragments of such orthologous peptides from other species of Chlamydia and peptides that are substantially similar to such peptides.
The present invention also teaches a method of vaccination comprising administering a vaccine formulated as described above to an animal either intravenously, intramuscularly, subcutaneously, by inhalation of a vapor or mist, or by inoculation in the form of a liquid, spray, ointment, pessary or pill into or onto the mucous membranes of the mouth, nose, lungs or urogenital tract or colon.
The methods of the invention may be practiced equally with human or non-human animal subjects.
The present invention also teaches a method of detecting Chlamydia infection-specific proteins produced by the Reticulate Body form of the organism. In this embodiment, antibodies raised to the infection-specific proteins are used in an immunological assay such as an Enzyme Linked Immunosorbant Assay or Biotin-Avidin assay or a radioimmunoassay or any other assay wherein specific antibodies are used to recognize a specific protein. Such assays may be used to detect both the quantity of proteins present and also the specificity of binding of such proteins. In such an assay, antibodies have attached to them, usually at the Fc portion, a detectable label, such as an enzyme, fluorescent marker, a radioactive marker or a Biotin-Avidin system marker that allows detection. A biological sample is provided from an animal that has been putatively exposed to Chlamydia. Such a sample may be, for example, whole blood, serum, tissue, saliva or a mucosal secretion. The sample is then contacted with the labeled antibody and specific binding, if any, is detected. Other methods of using infection-specific antibodies to detect infection-specific antigens that are present in cells or tissues include immunofluorescense, indirect-immunofluorescense and inmmunohistochemistry. In immunofluorescense, a fluorescent dye is bound directly to the antibody. In indirect-immunofluorescence, the dye is bound to an anti-immunoglobulin. Specific binding occurs between antigen and bound antibody is detected by virtue of flourescent emissions from the dye moiety. This technique would be particularly useful, for instance, for detection of Chlamydia antigen present on a urogenital mucosal smear.
Other techniques, such as competitive inhibition assays may also be used to assay for antigen, and one of ordinary skill in the art will readily appreciate that the precise methods disclosed may be modified or varied without departing from the subject or spirit of the invention taught herein.
The present invention also teaches a method of detection of Chlamydia infection-specific antibodies made against the Reticulate Body. In this embodiment a sample is provided from an animal putatively exposed to Chlamydia to determine whether the sample contains infection-specific antibodies. Such a sample may be, for example, whole blood, serum, tissue, saliva or a mucosal secretion. This sample is contacted with infection-specific antigens such that the amount and specificity of binding of the antibody may be measured by its binding to a specific antigen. Many techniques are commonly known in the art for the detection and quantification of antigen. Most commonly, the purified antigen will be bound to a substrate, the antibody of the sample will bind via its Fab portion to this antigen, the substrate will then be washed and a second, labeled antibody will then be added which will bind to the Fc portion of the antibody that is the subject of the assay. The second, labeled antibody will be species specific, i.e., if the serum is from a human, the second, labeled antibody will be anti-human-IgG antibody. The specimen will then be washed and the amount of the second, labeled antibody that has been bound will be detected and quantified by standard methods.
The present invention also teaches a method of treating a Chlamydial infection by directing a therapeutic agent against a specific target, such as: (i) an infection-specific protein of Chlamydia, (ii) a gene that encodes an infection-specific protein of Chlamydia and (iii) an RNA transcript that encodes an infection-specific protein of Chlamydia, wherein said therapeutic agent interacts with said target to affect a reduction in pathology.
For example, the present invention teaches a method of treating chlamydial infection wherein antisense technology is used to prevent the expression of infection-specific genes, thereby preventing the pathologies associated these proteins and preventing reproduction of the RB phase of Chlamydia. In this embodiment, RNA molecules complementary to transcripts of infection specific genes are introduced into the host cells that contain Chlamydia, and by binding to the mRNA transcripts of the infection-specific genes, prevent translation and therefore expression of the infection-specific proteins that are associated with pathogenesis.
The invention may be practiced to produce a vaccine against any species of Chlamydia, including C. psittaci, C. pecorum, C. trachomatis and C. pneumoniae. 